An annotated list of species of the Proteocephalus ... - Springer Link

Syst Parasitol (2007) 67:139–156 DOI 10.1007/s11230-006-9089-8

ORIGINAL PAPER

An annotated list of species of the Proteocephalus Weinland, 1858 aggregate sensu de Chambrier et al. (2004) (Cestoda: Proteocephalidea), parasites of fishes in the Palaearctic Region, their phylogenetic relationships and a key to their identification Toma´sˇ Scholz Æ Vladimı´ra Hanzelova´ Æ Andrea Sˇkerˇ´ıkova´ Æ Takeshi Shimazu Æ Leszek Rolbiecki Received: 21 March 2006 / Accepted: 28 June 2006 / Published online: 2 May 2007 Springer Science+Business Media B.V. 2007

Abstract A list and key to the identification of valid species of tapeworms of the Proteocephalus Weinland, 1858 aggregate sensu de Chambrier et al. (2004), i.e. species of the genus occurring in freshand brackish-water fishes in the Palaearctic Region, are provided, with data on their hosts and geographical distribution. Instead of 32 taxa listed by Schmidt (1986) and subsequent authors, only the following 14 species are considered to be valid: P. ambiguus (Dujardin, 1845) (type-species); P. cernuae (Gmelin, 1790); P. filicollis (Rudolphi, 1802); P. fluviatilis Bangham, 1925; P. gobiorum Dogiel & Bychowsky, 1939; P. longicollis (Zeder, 1800); P. macrocephalus (Creplin, 1825); P. midoriensis T. Scholz (&) A. Sˇkerˇ´ıkova´ Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Branisˇovska´ 31, 370 05 Ceske Budejovice, Czech Republic e-mail: [email protected] V. Hanzelova´ Parasitological Institute, Slovak Academy of Sciences, Hlinkova 3, 040 01 Kosˇice, Slovakia T. Shimazu Nagano Prefectural College, 8-49-7 Miwa, Nagano 380-8525, Japan L. Rolbiecki Department of Invertebrate Zoology, University of Gdansk, Al. Pilsudskiego 46, 81-378 Gdynia, Poland

Shimazu, 1990; P. percae (Mu¨ller, 1780); P. plecoglossi Yamaguti, 1934; P. sagittus (Grimm, 1872); P. tetrastomus (Rudolphi, 1810); P. thymalli (Annenkova-Chlopina, 1923); and P. torulosus (Batsch, 1786). An analysis of sequences of the nuclear genes (ITS2 and V4 region of 18S rDNA) revealed the following phylogenetic relationships for these taxa: P. torulosus ((P. midoriensis, P. sagittus) (P. fluviatilis (P. filicollis, P. gobiorum, P. macrocephalus)) (P. cernuae, P. plecoglossi, P. tetrastomus ((P. longicollis, P. percae) (P. ambiguus, P. thymalli)))). P. pronini Rusinek, 2001 from grayling Thymallus arcticus nigrescens is synonymised with P. thymalli. P. esocis La Rue, 1911 is apparently invalid but its conspecificity with either P. percae or P. longicollis could not be confirmed due to the absence of the scolex in the holotype and the unavailability of other material for morphological and molecular studies. P. osculatus (Goeze, 1782) has recently been transferred to Glanitaenia de Chambrier, Mariaux, Vaucher & Zehnder, 2004. The validity of the genus is supported by the position of G. osculata within the Proteocephalidea, based on molecular data, as well as its morphology and nature of the definitive host (the European wels Silurus glanis). P. hemispherous Rahemo & Al-Niaeemi, 2001, described from S. glanis in Iraq, is transferred to Postgangesia Akhmerov, 1960 as Postgangesia hemispherous (Rahemo & Al-Niaeemi, 2001) n. comb.

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140

Syst Parasitol (2007) 67:139–156

Introduction

been synonymised and all taxa from European fishes were revised and redescribed (Scholz & Hanzelova´, 1998). In order to avoid confusions regarding the validity of species of the Proteocephalus aggregate, an annotated list of its taxa considered to be valid is presented, with a brief information on their definitive hosts and geographical distribution. Phylogenetic relationships of all valid taxa inferred from an analysis of the ITS2 and V4 regions of 18S rRNA genes are also discussed, based on the majority consensus of 32 trees of maximum parsimony analysis of three matrices (Fig. 1).

Tapeworms of the Proteocephalus Weinland, 1858 aggregate sensu de Chambrier et al. (2004) are parasitic in freshwater fishes in the Palaearctic Region (see de Chambrier, Zehnder, Vaucher & Mariaux, 2004). Since 1780, when O.F. Mu¨ller described Taenia percae, the first species currently recognised as a member of Proteocephalus, several tens of taxa (73 according to Freze, 1965 and 91 according to Schmidt, 1986) have been described. However, recent studies on the systematics of species of the aggregate and several taxa of Proteocephalus (sensu lato) from North American freshwater fishes, carried out by the present authors (Scholz & Hanzelova´, 1998, 1999; Hanzelova´ & Scholz, 1999), have demonstrated that the actual number of valid species of Proteocephalus is much lower. In a series of papers, numerous species have

Fig. 1 Phylogenetic relationships of Proteocephalus species from the Palaearctic Region based on 5.8S + ITS2 and V4 region of 18S rRNA gene sequences: majority consensus tree of 32 consensus trees obtained by maximum parsimony analyses (according to the matrix alignment parameters: TL = 15363041; CI = 0.75-0.78). The numbers at the nodes show bootstrap support

Materials and methods The list is based primarily on the results of multidisciplinary studies of the present authors and their co-workers: Shimazu (1990, 1993), Hanzelova´ &

Acanthotaenia sp. 92

Gangesia parasiluri Silurotaenia siluri

100

Paraproteocephalus parasiluri Glanitaenia osculata Proteocephalus torulosus

100 100

Proteocephalus midoriensis Proteocephalus sagittus

99

Proteocephalus fluviatilis 100

Proteocephalus macrocephalus 100

Proteocephalus filicollis Proteocephalus gobiorum

96

Proteocephalus cernuae Proteocephalus plecoglossi Proteocephalus tetrastomus

100

Proteocephalus longicollis1 50

Proteocephalus longicollis2 Proteocephalus percae

56 100

Proteocephalus ambiguus Proteocephalus thymalli

123


Scholz (1992, 1993, 1999), Hanzelova´ & Sˇpakulova´ (1992), Sˇpakulova´ & Hanzelova´ (1992), Scholz & Hanzelova´ (1994, 1998, 1999), Sˇna´bel, Hanzelova´ & Fagerholm (1994), Sˇna´bel, Hanzelova´, Mattiucci, D’Amelio & Paggi (1996), Hanzelova´, Scholz & Fagerholm (1995), Hanzelova´, Sˇna´bel, Sˇpakulova´, Fagerholm & Kra´l’ova´ (1995), Hanzelova´, Sˇna´bel & Sˇpakulova´ (1996), Hanzelova´, Sˇna´bel, Kra´l’ova´, Scholz & D’Amelio (1999), Scholz, Hanzelova´ & Sˇna´bel (1995), Scholz, Sˇpakulova´, Sˇna´bel, Kra´l’ova´ & Hanzelova´ (1997), Scholz, Dra´bek & Hanzelova´ (1998), Scholz, Hanzelova´, Kra´lova´ & Griffiths (1998), Scholz, Sˇkerˇ´ıkova´, Hanzelova´, Koubkova´ & Barusˇ (2003), Scholz, Marcogliese, Bourque, Sˇkerˇ´ıkova´ & Dodson (2004), Turcˇekova´ & Kra´l’ova´ (1995), Kra´l’ova´ (1996), Kra´l’ova´ & Sˇpakulova´ (1996), Sˇkerı´kova´, Hypsˇa & Scholz (2001) and Hypsˇa, Sˇkerˇ´ıkova´ & Scholz (2005). The most detailed information on the morphology of European taxa of Proteocephalus, including key to the species, was provided by Scholz & Hanzelova´ (1998), whereas Japanese taxa were redescribed by Shimazu (1990). Scholz, Dra´bek & Hanzelova´ (1998) presented a detailed account of the morphology and measurements of the scoleces of most species of Proteocephalus from the Palaearctic Region. In addition to Proteocephalus species studied previously, new material of P. ambiguus (Dujardin, 1845) from Pungitius pungitius (L.) in Poland and the following species from Japan and Finland was morphologically evaluated: Japan: P. fluviatilis Bangham, 1925 from Micropterus dolomieu Lace´pe`de, Lake Nojiri, Nagano Prefecture; P. midoriensis Shimazu, 1990 from Lefua echigonia Jordan & Richardson, Iiyama, Nagano Prefecture and the Yanamunegawa River, Kosei Town, Shiga Prefecture; P. plecoglossi Yamaguti, 1934 from Plecoglossus altivelis altivelis (Temminck & Schlegel), Lake Biwa, Shiga Prefecture; P. plecoglossi juv. from Gymnogobius isaza (Tanaka), Hemibarbus barbus (Temminck & Schlegel), Opsariichthys uncirostris (Temminck & Schlegel) and Tridentiger brevispinis Katsuyama, Arai & Nakamura, Lake Biwa, Shiga Prefecture; P. tetrastomus (Rudolphi, 1810) from Hypomesus nipponensis (McAllister), Lake Suwa, Nagano Prefecture, and Lake Ogawara, Aomori Prefecture; Finland: P. cernuae (Gmelin, 1790) from Gymnocephalus cernuus (L.); P. filicollis (Rudolphi, 1802) from Gasterosteus aculeatus L.; P. longicollis

141

(Zeder, 1800) from Coregonus albula (L.) and C. widegreni Malmgren; P. percae (Mu¨ller, 1780) from Perca fluviatilis L.; P. tetrastomus from Osmerus eperlanus (L.) – all cestodes from the Bothnian Bay at Kiviniemi near Oulu, Finland. An assessment of the phylogenetic relationships is primarily based on the methodology and multigene analysis carried out by Hypsˇa et al. (2005), but molecular data provided by Zehnder & Mariaux (1999), Sˇkerˇ´ıkova´ et al. (2001), de Chambrier et al. (2004) and Scholz et al. (2004) were also considered, if necessary. In addition, six new sequences of the V4 region of 18S rRNA gene and/or 5.8S + ITS-2 of P. ambiguus from Pungitius pungitius in Poland, P. longicollis (Zeder, 1800) from Oncorhynchus mykiss (Walbaum) in Scotland, P. percae from Perca fluviatilis L. in Switzerland and P. thymalli from Thymallus arcticus (Pallas) in Russia, were obtained and analysed (Table 1). Cestodes intended for sequencing were fixed with 96% ethanol. Total DNA was extracted from 0.5 cm of strobila using the QIAamp Tissue kit (Qiagen). The V4 region of the 18S rRNA gene was amplified by PCR using the primers Ces1 (5’-CCA GCA GCC GCG GTA ACT CCA-3’) and Ces2 (5’-CCC CCG CCT GTC TCT TTT GAT-3’) designed according to the complete sequences of the 18S rRNA gene of Proteocephalus exiguus La Rue, 1911 (= Proteocephalus longicollis (Zeder, 1800); Kra´l’ova´ et al., 1997). The 5.8S rRNA-ITS2 genes were amplified by PCR using the primers Proteo1 (5’-CGG TGG ATC ACT CGG CTC-3’) and Proteo2 (5’-TCC TCC GCT TAT TGA TAT GC-3’) designed according to the complete sequences of the ITS1-ITS2 genes of Eubothrium crassum (Bloch, 1779) and E. salvelini (Schrank, 1790) (Kra´l’ova´, Hanzelova´, Scholz, Gerdeaux & Sˇpakulova´, 2001). The following schedule was used for the PCR: first, 15 min at 958C (HotStarTaqTm DNA polymerase); then 30 cycles: denaturation, 1 min at 948C, annealing 1 min at 608C, extension, 2 min at 728C, and at the end the final extension 10 min at 688C. The PCR products were cloned into pGEM-T Easy System 1 (Promega) and sequenced in both directions using T7 and SP6 primers. DNA sequencing was performed on automated sequencer model 310 ABI PRISM (PE-Biosystems) using BigDye DNA sequencing kit (PE-Biosystems). Accession numbers of the six sequences obtained (DQ427096–DQ427101), as well as the 32 sequences

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142 Table 1 The taxa and genes included in the phylogenetic analysis (new sequences in bold and marked with an asterisk)


Sample

ITS-2 + 5.8S

V4-18S rRNA

Acanthotaenia sp. (outgroup)

AY551137

AF267292

Gangesia parasiluri (outgroup)

AY551144

AF267293

Glanitaenia osculata

AY551169

AF335508

Paraproteocephalus parasiluri

AY551157

AY551121

Proteocephalus ambiguus

DQ427096*

DQ427100*

Proteocephalus cernuae

AY551160

AY551125

Proteocephalus filicollis

AY551162

AF335506

Proteocephalus fluviatilis Proteocephalus gobiorum

AY551163 no PCR product

AY551126 AY551127

Proteoceph. longicollis 1 (Switzerland)

AY551166

X99976

Proteoceph. longicollis 2 (Scotland)

DQ427097*

DQ427101*

Proteocephalus macrocephalus

AY551167

AF335507

Proteocephalus midoriensis

AY551168

AY551130

Proteocephalus percae

DQ427098*

AF335509

Proteocephalus plecoglossi

AY551171

AY551132

Proteocephalus sagittus

AY375548

AY375550

Proteocephalus tetrastomus

AY379113

AF335510

Proteocephalus thymalli

DQ427099*

no PCR product

Proteocephalus torulosus

AY375549

AF335511

Silurotaenia siluri (outgroup)

AY551175

AF267297

retrieved from Gene Bank, are given in Table 1. Three taxa, Acanthotaenia sp., Gangesia parasiluri Yamaguti, 1934 and Silurotaenia siluri (Batsch, 1786), were used as outgroups, based on their position among proteocephalideans (de Chambrier et al., 2004; Hypsˇa et al., 2005). The new sequences were aligned manually to the three alignments combining ITS2 + V4-18S rRNA (‘‘basic matrices’’ used by Hypsˇa et al., 2005) using the Bioedit program (Hall, 1999) in respect of their Table 2 Alignment parameters

high similarity to the sequence of P. longicollis from Coregonus lavaretusin Switzerland (see alignment parameters in Table 2). The alignments are available upon request from the authors. The phylogenetic analysis and calculation of nodal support (maximum parsimony – MP and bootstrap) were performed using PAUP version 4.0 (Swofford, 2003). The MP analyses were performed under the assumption of Tv/Ts ratio 1:1, 1:2 and 1:3. A bootstrap support (1,000 replicates) was calculated for Tv/Ts 1:2.

Matrix 1

Matrix 2

Matrix 3

Gap opening penalty

15

1

0.7

Gap extension penalty

10

0.1

0.7

Transition weight

20

1

0.7

Number of characters

1,907

2,129

1,882

Alignment parameters:

Phylogenetic analyses:

123

Tv/Ts ratio

1:1

1:2

1:3

1:1

1:2

1:3

1:1

1:2

1:3

Informative characters Number of trees

413 2

440 4

440 2

391 2

409 4

409 2

424 10

448 4

448 2


Reference material (types or vouchers) of P. pamirensis Dzhalilov & Ashurova, 1971 (syn. of P. sagittus – see Scholz et al., 2003) and P. pronini Rusinek, 2001 has not been available, despite several attempts to contact the authors of the original descriptions or institution where the type-specimens should have been deposited. These two taxa and specimens of P. esocis were not available for molecular analysis. In the present list, information on the type- and other hosts (only reliable host records are considered; for a more complete list of hosts from Europe – see Scholz & Hanzelova´, 1998), type-localities and geographical distribution is provided. References in the list of valid species refer only to the most pertinent papers, especially those providing detailed data on the morphology and systematics of the respective taxa. Molecular data and phylogenetic analyses New sequences of variable V4 region of 18S rRNA gene and complete ITS2 were obtained for 2 and 4 taxa, respectively (Table 1). The lengths of V4-18S rRNA varied from 454 bp (P. ambiguus) to 455 bp (P. longicollis), and for ITS2 from 741 bp (P. ambiguus) to 745 bp (P. longicollis). The three matrices, combining the ITS2 and V418S rRNA gene sequences, provided altogether 1,882-2,129 characters (Table 2). Under MP criterion, the number of parsimony informative characters varied from 391-448 (Table 2). The majority consensus of 32 trees yielded a partly resolved topology of three well-supported monophyletic clusters with P. torulosus at their base (Fig. 1).

List of valid species Proteocephalus ambiguus (Dujardin, 1845) – type-species Syns Taenia ambigua Dujardin, 1845; Proteocephalus filicollis (Rudolphi, 1802) auct. in part Type-host: Nine-spined stickleback Pungitius pungitius (L.) (Gasterosteiformes: Gasterosteidae). Type-locality: Rennes, France. Distribution: Europe, including Russia. Reference: Rødland (1983); Scholz & Hanzelova´ (1998).

143

Comments: P. ambiguus occurs exclusively in the nine-spined stickleback (Willemse, 1968). Many confusions have existed as to the differentiation of this species from morphologically similar species, P. filicollis, a parasite of the three-spined stickleback Gasterosteus aculeatus, until Willemse (1968) and Rødland (1983) confirmed the validity of both the taxa and distinguished them on the basis of several morphological and biological characteristics. Although Scholz & Hanzelova´ (1998) questioned some of the differential features provided by Rødland (1983), they considered both the species to be valid. The validity of the species is supported by differences in the sequences of the ITS2 and 18S rRNA from those of P. filicollis (67.9% and 98.0%, respectively; see Table 3). Sequences of ITS-2 + 5.8S of P. ambiguus and P. thymalli were almost identical (similarity of 99.9%), which contrasts with the fact that these taxa are markedly different in their morphology (see Scholz & Hanzelova´, 1998 and a key provided below). A high similarity (99.1%) was also found between the sequence of the V4 region of the 18S rRNA gene of P. ambiguus and that of one of two samples of P. longicollis (Table 3), although the latter species closely resembles P. thymalli in its morphology, thus being markedly different from P. ambiguus. The reasons for this surprising genetic similarity of morphologically distinct taxa specific to unrelated fish hosts require further study. Phylogenetic relationships: P. ambiguus formed a clade with morphologically distinct P. thymalli (Fig. 1) and similarity of their ITS2 sequences was very high (99.9%) (no PCR product of 18S rDNA was obtained from P. thymalli sample despite several attempts). Correctness of P. ambiguussequences was confirmed by repeated sequencing, with the result differing only in 2 nucleotides for both P.ambiguus samples; species identification of P. ambiguus samples sequenced was also confirmed before repeated trials. Proteocephalus cernuae (Gmelin, 1790) Syn. Taenia cernuae Gmelin, 1790 Type-host: Ruff Gymnocephalus cernuus (L.) (Perciformes: Percidae). Type-locality: Not known, probably Germany. Distribution: Europe, northern Asia (Russia).

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144


Table 3 Percent similarity of individual sequences 1. 1. Proteocephalus ambiguus

*

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

99.8 98.0 98.5 98.2 96.7 99.1 97.8 96.7 94.5 99.3 99.3 98.0

2. Proteocephalus cernuae

95.2

3. Proteocephalus filicollis 4. Proteocephalus fluviatilis

67.9 67.6 * 97.8 99.3 95.2 97.6 98.5 95.9 93.2 97.8 97.8 97.1 74.4 73.5 74.4 * 98.0 95.9 98.0 97.6 96.1 93.6 98.2 98.2 97.4

5. Proteocephalus gobiorum

-

*

-

14.

15.

16.

-

96.7

-

96.9

98.2 98.7

-

95.6 96.0

98.5

-

95.8

96.9

-

93.9

99.3

-

96.3

98.0

-

95.4

99.6

98.2 98.7 98.5 96.9 99.3 98.0 96.9 94.3 99.6 99.6 98.2 100

-

-

*

95.4 97.8 99.1 95.9 93.4 98.0 98.0 97.1

6. Proteocephalus longicollis 1

97.7 95.6 68.1 74.1

-

*

96.3 95.2 94.2 92.0 96.5 96.5 95.2

7. Proteocephalus longicollis 2

98.1 95.5 67.1 74.1

-

99.3

8. Proteocephalus macrocephalus 64.2 65.8 83.8 72.0

-

64.6 64.5

*

97.4 96.3 93.7 98.9 98.9 97.6 *

95.4 93.0 97.6 97.6 96.7

9. Proteocephalus midoriensis

69.2 68.9 60.6 64.7

-

69.6 69.7 59.9

10. Proteocephalus osculatus#

66.6 69.9 57.1 56.3

-

66.8 67.1 59.6 58.8

*

93.3 96.5 96.5 98.3

11. Proteocephalus percae

97.4 94.4 67.9 73.9

-

97.5 97.6 64.2 69.2 64.7

12. Proteocephalus plecoglossi

95.4 93.0 67.9 73.7

-

95.1 95.4 65.7 68.8 66.5 94.3

13. Proteocephalus sagittus

68.7 67.4 61.9 50.9

-

68.5 69.0 60.4 81.3 67.7 68.4 68.1

14. Proteocephalus tetrastomus

96.0 94.2 67.5 60.7

-

95.9 96.0 63.7 69.2 66.2 94.8 94.9 68.5

15. Proteocephalus thymalli

99.9 95.1 67.8 74.2

-

97.6 98.0 64.3 69.2 66.5 97.3 95.2 68.0

16. Proteocephalus torulosus

72.2 71.8 64.2 67.0

-

72.0 72.4 63.6 68.2 70.1 71.9 72.0 70.3

72.8 72.2

*

93.9 93.9 94.3 *

99.1 97.8 *

96.9

-

95.2

94.3

-

92.8

99.6

-

96.5

97.8

99.6

-

96.5

*

98.2

-

96.9

*

-

96.9

95.9

*

*

* ITS-2 + 5.8S similarities on the left from the asterisks, V4-18S rRNA similarities on the right #

Synonym of Glanitaenia osculata (see de Chambrier et al., 2004)

Reference: Scholz & Hanzelova´ (1998). Comments: This species seems to be a specific parasite of ruff. Findings in other percids (Perca fluviatilis and Sander lucioperca) should be confirmed on the basis of new material. The species was redescribed by Scholz and Hanzelova´ (1998), who provided morphological characters enabling its differentiation from P. percae and other taxa of the genus. Phylogenetic relationships: In a multigene analysis (Hypsˇa et al., 2005), P. cernuae formed a clade with P. longicollis from salmoniform fish, although the species differ considerably in scolex morphology (Scholz, Dra´bek, & Hanzelova´, 1998) and several strobilar characters (Scholz & Hanzelova´, 1998). The adding of new sequences of P. ambiguus and P. thymalli, however, changed the topology of the tree and P. cernuae appeared in a polytomy formed by two species from osmeriform fish, namely P. plecoglossi and P. tetrastomus (Fig. 1). In all analyses, P. cernuae was not closely related to P. percae, another species from percid fish, but sequence similarity was relatively high (94.4% for ITS2 and 99.6% for V4 18S rDNA).

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Proteocephalus filicollis (Rudolphi, 1802) Syn. Taenia filicollis Rudolphi, 1802 Type-host: Three-spined stickleback Gasterosteus aculeatus L. (Gasterosteiformes: Gasterosteidae). Type-locality: Greifswald, Germany. Distribution: Circumboreal (Europe, northern Russia, North America). References: Rødland (1983); Scholz & Hanzelova´ (1998). Comments: This cestode is specific to three-spined stickleback in the Holarctic Region (Freze, 1965; Hoffman, 1999). Phylogenetic relationships: Close relationship of P. filicollis with P. macrocephalus from eels was found in all analyses based on morphological data, 18S (Sˇkerˇ´ıkova´ et al., 2001) and 28S rRNA (de Chambrier et al., 2004; Zehnder & Mariaux, 1999). In a multigene analysis (Hypsˇa et al., 2005; present data), these two species formed a clade together with P. gobiorum from gobiids. Sequence similarity of these cestodes (Table 2) corresponds neither with relationships of their definitive hosts, stickleback, eels and gobiid fishes, because they are phylogenetically


distant (Nelson, 1994), nor with their morphology, although the ‘‘wide’’ form of P. filicollis somewhat resembles in its strobilar morphology that of P. macrocephalus (see Scholz & Hanzelova´, 1998).

Proteocephalus fluviatilis Bangham, 1925 Syn. Proteocephalus osburni Bangham, 1925 Type-host: Smallmouth bass Micropterus dolomieu Lace´pe`de (Perciformes: Centrarchidae). Type-locality: Ohio, USA. Other hosts: Ambloplites rupestris (Rafinesque), Lepomis auritus (L.), Micropterus salmoides (Lace´pe`de) (Centrarchidae). Distribution: North America, Japan (introduced). Reference: Shimazu (1990). Comments: Proteocephalus fluviatilis was described from the smallmouth bass Micropterus dolomieu and later reported from other centrarchids in North America (Hoffman, 1999; Freze, 1965; Margolis & Arthur, 1979). Shimazu (1990) found this cestode in the largemouth bass M. salmoides from Lake Kizaki, Nagano Prefecture, in central Japan. The present material of P. fluviatilis was found in smallmouth bass from Lake Nojiri, Nagano Prefecture. The parasite was introduced into Japan, most probably with centrarchid fishes imported from North America. Phylogenetic relationships: In a multigene analysis (Hypsˇa et al., 2005; present study), P. fluviatilis formed a sister group to the clade composed of P. gobiorum, P. filicollis and P. macrocephalus. All these taxa can be easily distinguished each other on the basis of their scolex morphology (Scholz, Dra´bek, & Hanzelova´, 1998) and some characteristics of the strobila (Scholz & Hanzelova´, 1998; Shimazu, 1990). Sequence similarity of P. fluviatilis with two other species from perciform fishes, i.e. P. cernuae and P. percae, is rather low (73.5–73.9% for ITS2 and 98.2–98.7% for V4). Proteocephalus gobiorum Dogiel & Bychowsky, 1939 Syn. Proteocephalus subtilis Naidenova, 1974 Type-host: Not designated, probably Benthophilus macrocephalus (Pallas) (Perciformes: Gobiidae). Type-locality: Volga River estuary, Caspian Sea, Russia.

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Other hosts: Gobies (Benthophilus, Gobius, Neogobius, Pomatoschistus). Distribution: Northern Europe, Russia and Ukraine. Reference: Scholz & Hanzelova´ (1998). Comments: This tapeworm appears to be a specific parasite of gobies (Gobiidae) that have been reported in brackish waters of the Eastern Atlantic (Skagerrak), Baltic, Black and Caspian Seas. Scholz & Hanzelova´ (1998) did not find any morphological differences between specimens from Gobius niger and Pomatoschistus microps from Norway and those from a gobiid fish from Russia, probably from the Caspian Sea. However, the material of P. gobiorum studied by Scholz & Hanzelova´ (1998) was limited in number and no specimens were available for a comparative DNA analysis of tapeworms from different fish hosts and geographical regions. Phylogenetic relationships: Molecular data demonstrate a close relationship of this species with P. filicollis and P. macrocephalus (Fig. 1), despite the unrelatedness of their fish definitive hosts and marked differences in their morphology, especially the scolex (P. gobiorum is devoid of an apical sucker which is present, yet reduced, in P. filicollis and P. macrocephalus). Despite several attempts, no PCR product for the ITS2 gene of P. gobiorum was obtained and thus its close relatedness to P. filicollis and P. macrocephalus should be confirmed by other gene(s). The absence of ethanol-fixed material of P. gobiorum from different seas and gobiid fish also makes it impossible to assess the range of intraspecific variability of this taxon, with its fairly wide host spectrum and distribution.

Proteocephalus longicollis (Zeder, 1800) Syns Alyselminthus longicollis Zeder, 1800; Taenia salmonisomul Pallas, 1811; T. cyclops Linstow, 1877; T. salmonisumblae Zschokke, 1884; T. salvelini Linton, 1897 (?); Ichthyotaenia esocis Schneider, 1905 (?); I. agonis Barbieri, 1909; Proteocephalus pusillus Ward, 1910; P. exiguus La Rue, 1911; P. fallax La Rue, 1 911; P . neglectus La Rue, 1 911; P. laruei Faust, 1920; P. arcticus Cooper, 1921; P. coregoni Wardle, 1932 (?); P. wickliffi Hunter & Bangham, 1933; P. parallacticus MacLulich, 1943; P. californicus Haderlie, 1950; P. salmonidicola Alexander, 1951; P. pollanicola Gresson, 1952; P. tumidocollis Wagner, 1953; P. albulae Freze & Kazakov, 1969

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Type-host: Trout Salmo trutta trutta L. (Salmoniformes: Salmonidae). Type-locality: Not known, probably Germany. Other hosts: Salmoniform fishes (Salmonidae: Oncorhynchus, Salmo, Salvelinus; Coregonidae: Coregonus) and smelt (Osmeriformes: Osmeridae). Distribution: Europe, Asia (Armenia, Russia, Mongolia), North America. References: Hanzelova´ & Scholz (1993, 1999); Hanzelova´, Scholz & Fagerholm (1995); Hanzelova´ et al. (1996); Scholz, Dra´bek & Hanzelova´ (1998), Scholz, Hanzelova´ et al. (1998); Scholz & Hanzelova´ (1998). Comments: This is the most variable and frequent species of Proteocephalus that occurs in a wide range of definitive hosts, especially trout and whitefish, in the Holarctic Region (Ieshko & Anikieva, 1980; Hanzelova´ & Scholz, 1999; Scholz & Hanzelova´, 1998). Such a wide host range may explain the fact that sequences of the two isolates of P. longicollis from different hosts differed (Table 3), even more than some morphologically well delimited taxa specific to unrelated hosts. However, despite these sequence differences, especially those in the V4 region of 18S rRNA, both isolates are considered to represent one species, because they appeared most closely to each other on the phylogenetic tree, forming a monophyletic group with P. percae (Fig. 1), and they share morphological characteristics considered to be species-specific, such as the shape of the scolex and apical sucker, and the morphology of the terminal genitalia formed by a long cirrus-sac and vaginal canal with a well-developed vaginal sphincter (Hanzelova´, Sˇna´bel et al., 1995; Scholz & Hanzelova´, 1998). Only a detailed molecular study based on multigene analysis of a high number of samples from a much wider spectrum of fish hosts might reveal possible existence of cryptic species (Olson & Tkach, 2005). Phylogenetic relationships: P. longicollis is morphologically similar to P. percae from percid fish (see Sˇna´bel et al. 1994; Hanzelova´ , Sˇna´bel, Sˇpakulova´, et al., 1995; Scholz et al., 1995; Kra´l’ova´, 1996). This tallies with results of phylogenetic analyses based on sequences of 16S rDNA (Zehnder & Mariaux, 1999 – fig. 2), 18S rRNA (Sˇkerˇl’ı´kova´ et al. 2001 – fig. 1; this study) and ITS2 (present data – Fig. 1) genes as well as morphological characters (Sˇkerˇ´ıkova´ et al., 2001 – fig. 2). The sequence similarity of these species, especially that of ITS2, is high (97.5-97.6% – Table 2).

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Proteocephalus macrocephalus (Creplin, 1825) Syns Taenia macrocephala Creplin, 1825; T. hemisphaerica Molin, 1859; T. dilatata Linton, 1889 Type-host: Common eel Anguilla anguilla (L.) (Anguilliformes: Anguillidae). Type-locality: Greifswald, Germany. Other hosts: Eels (Anguilla spp.). Distribution: Europe, including Russia, North Africa (Morocco), North America. References: Scholz et al. (1997); Scholz & Hanzelova´ (1998); Hoffman (1999). Comments: This cestode is specific to eels and has circumboreal (Holarctic) distribution (Freze, 1965; Scholz & Hanzelova´, 1998; Hoffman, 1999). Phylogenetic relationships: Results of analyses inferred from morphological characters and molecular data (Zehnder & Mariaux, 1999; Sˇkerˇ´ıkova´ et al., 2001; de Chambrier et al., 2004) are congruent in demonstrating a close relationship of P. macrocephalus with P. filicollis. These taxa also form a wellsupported clade with P. gobiorum (Fig. 1), with very high similarity of sequences of the V4 region of 18S rRNA (99.1-99.3%).

Proteocephalus midoriensis Shimazu, 1990 Type-host: Lefua echigonia Jordan & Richardson (Cypriniformes: Balitoridae). Type-locality: Midori, Nagano Prefecture, Japan. Distribution: Japan. Reference: Shimazu (1990). Comments: Proteocephalus midoriensis has been found only in the type-host and its distribution area may be restricted to central Japan. Morphologically, it is rather similar to P. sagittus (Grimm, 1870), another cestode from a balitorid fish, Barbatula barbatula (L.). P. midoriensis differs in having a smaller scolex, more testes arranged in at least two layers, a less convoluted seminal vesicle, a smaller cirrus-sac, deeply indented ovarian lobes, a much more weakly developed vaginal sphincter, a longer and more sinuous seminal receptacle, more uterine branches and larger eggs (Shimazu, 1990). Moreover, no worms of Proteocephalus have been obtained from B. barbatulain Hokkaido, northern Japan (T. Shimazu, unpublished data).


Phylogenetic relationships: Based on molecular data (Hypsˇa et al., 2005; this study), P. midoriensis and P. sagittus form a well-supported clade. Values of sequence similarity (81.3% and 98.3% for ITS2 and V4, respectively) indicate that both taxa are separate species differing slightly in strobilar morphology, the spectrum of definitive hosts and geographical distribution. Proteocephalus percae (Mu¨ller, 1780) Syns Taenia percae Mu¨ller, 1780; T. ocellata Rudolphi, 1802; Ichthyotaenia esocis Schneider, 1905 (?); P. dubius La Rue, 1911 Type-host: Perch Perca fluviatilis L. (Perciformes: Percidae). Type-locality: Denmark. Other hosts: Aspro, Gymnocephalus, Sander (Percidae). Distribution: Europe, Asia (Azerbaidzhan, Kazakhstan, Mongolia, Siberia). References: Scholz & Hanzelova´ (1998), Hanzelova´ et al. (1999). Comments: Proteocephalus percae is a common and widely distributed parasite of perch, which may also occur in other percid fishes (Freze, 1965; Anikieva, 1995a; Scholz & Hanzelova´, 1998). Phylogenetic relationships: Morphological and molecular data (sequences of ITS, 16S and 18S rRNA genes – Zehnder & Mariaux, 1999; Sˇkerˇ´ıkova´ et al., 2001; present study) demonstrate a close phylogenetic relationship of P. percae with P. longicollis (Fig. 1). This corresponds well with the morphological similarity of these species (Hanzelova´, Sˇna´bel et al., 1995, 1999), but does not reflect phylogenetically unrelatedness of their fish definitive hosts (Nelson, 1994). Zehnder & Mariaux (1999) found P. percae to be closely related to P. torulosus, but this conclusion was based on the incorrect partial sequence of 28S rRNA of P. percae (M.P. Zehnder – personal communication). de Chambrier et al. (2004) did not include P. torulosus in their analysis, but P. percae appeared as a basal taxon of the Palaearctic species of Proteocephalus, i.e. in the same position as P. torulosus in a tree inferred from a multigene analysis of several nuclear genes performed by Hypsˇa et al. (2005). It is probable that the sequence belongs in fact to P. torulosus.

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Proteocephalus plecoglossi Yamaguti, 1934 Syn. Proteocephalus neglectus of Kataoka & Momma (1932) Type-host: Plecoglossus altivelis altivelis (Temminck & Schlegel) (Osmeriformes: Plecoglossidae). Type-locality: Lake Biwa, Shiga Prefecture, Japan. Distribution: Japan. References: Yamaguti (1934); Shimazu (1990, 1993). Comments: Proteocephalus plecoglossi is a specific parasite of Plecoglossus altivelis altivelis and occurs only in Lake Biwa, central Japan (Kataoka & Momma, 1932; Yamaguti, 1934; Shimazu, 1990, 1993). This cestode resembles in its morphology Proteocephalus neglectus La Rue, 1911 (= P. longicollis, according to Scholz & Hanzelova´, 1998) and was misidentified as P. neglectus by Kataoka & Momma (1932). Phylogenetic relationships: Molecular data also indicate close relationships of P. plecoglossi with P. longicollis (Zehnder & Mariaux, 1999; Hypsˇa et al., 2005; this study – Fig. 1, Table 3). However, in an analysis of sequences of ITS and the V4 region of 18S rRNA genes, P. plecoglossi appeared in a polytomy composed of P. tetrastomus and P. cernuae (Fig. 1). P. tetrastomus also occurs in osmeriform fish, but it differs markedly from P. plecoglossi in its morphology, especially in possessing trapezoid, well-separated proglottids and a rudimentary, almost indistinguishable apical sucker (versus the strobila composed of rectangular or oblong proglottids and a reduced but readily distinguishable apical sucker in P. plecoglossi) (Shimazu, 1990, 1993; Scholz & Hanzelova´, 1998).

Proteocephalus sagittus (Grimm, 1872) Syns Taenia sagitta Grimm, 1872; Proteocephalus pamirensis Dzhalilov & Ashurova, 1971 Type-host: Stone loach Barbatula barbatula (L.) (Cypriniformes: Balitoridae). Type-locality: St. Petersburg, Russia. Other hosts: Noemacheilus stoliczkai (Steindachner) [syn. of Tryplophysa stoliczkae (Steindachner)] (Balitoridae), Cobitis taenia L. (Cypriniformes: Cobitidae). Distribution: Europe, including Russia and the Ukraine, Tadjikistan. Reference: Scholz et al. (2003).

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Comments: Scholz & Hanzelova´ (1998) synonymised P. sagittus with P. torulosus (Batsch, 1786), a parasite of cyprinid fishes in the Holarctic Region. However, Scholz et al. (2003) resurrected the former taxon on the basis of new morphological and molecular data obtained during a study of freshly collected material of P. sagittus from the type-host. P. sagittus differs from P. torulosus in scolex morphology, especially its shape, the position of the vagina in relation to the cirrus-sac, the length of the cirrus-sac, more distinct osmoregulatory canals and sequences of the V4 region and ITS2 (sequence similarity 96.9% and 70.3%, respectively) (Scholz et al., 2003; Table 2). The authors (Scholz et al., 2003) also synonymised P. pamirensis, a species described from the Tibetan stone loach Tryplophysa stoliczkae (Steindachner) in Tadjikistan (Dzhalilov & Ashurova, 1971), with P. sagittus. Phylogenetic relationships: P. sagittus forms a wellsupported clade with another Proteocephalus species, P. midoriensis, from a balitorid fish limited in distribution to central Japan. A combined analysis (Hypsˇa et al., 2005; present data – Fig. 1) has also shown that P. torulosus and P. sagittus actually represent two separate, relatively unrelated species, thus confirming the taxonomic conclusions of Scholz et al. (2003). Proteocephalus tetrastomus (Rudolphi, 1810) Syns Scolex tetrastomus Rudolphi, 1810; Taenia longicollis of Linstow (1891), Dubinina (1952, 1987) and Freze (1965) [nec Zeder (1800)] Type-host: Not known. Type-locality: Not known, probably Germany. Other hosts: Hypomesus nipponensis (McAllister), Osmerus eperlanus (L.) (probably type-host), O. mordax (Mitchill) (all Osmeriformes: Osmeridae). Distribution: Europe, including Russia, Japan, Canada. References: Willemse (1969); Shimazu (1990); Anikieva (1998); Scholz & Hanzelova´ (1998); Scholz et al. (2004). Comments: This species had been confused with P. longicollis by many authors, e.g. Linstow (1891), Dubinina (1952), Freze (1965), etc., until Willemse (1969) recognised the presence of two, morphologically and ecologically distinct species of

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Proteocephalus in Osmerus eperlanus, namely P. tetrastomus and P. longicollis. The former species is a specific parasite of smelts (Osmeridae) recorded from the northern part of Europe, Russia and Japan. The latter taxon, P. longicollis, is a very rare parasite of smelt but occurs frequently in numerous salmonid and coregonid fish (Scholz & Hanzelova´, 1998; Hanzelova´ & Scholz, 1999). Recently, P. tetrastomus has been found in larval rainbow smelt (O. mordax) in the Saint Lawrence estuary, Canada (Scholz et al., 2004), where it is supposed to regulate the early lifehistory survival of rainbow smelt estuarine populations (Bourque et al., 2006). Phylogenetic relationships: Morphological (Sˇkerˇ´ıkova´ et al., 2001) and molecular data (Zehnder & Mariaux, 1999; Hypsˇa et al., 2005; present study – Fig. 1) demonstrate consistently a close phylogenetic relationships of P. tetrastomus with P. plecoglossi that form a polytomy with P. cernuae (Fig. 1). Both the taxa from osmeriform fish, P. tetrastomus and P. plecoglossi, are easy to distinguish on the basis of their morphology (see Shimazu, 1990 and comments above on P. plecoglossi). Proteocephalus thymalli (Annenkova-Chlopina, 1923) Syns Ichthyotaenia thymalli Annenkova-Chlopina, 1923; Proteocephalus pronini Rusinek, 2001 (new synonym) Type-host: Baikal black grayling Thymallus arcticus baicalensis (Dybowski) (Salmoniformes: Salmonidae). Type-locality: Lake Baikal, Russia. Other hosts: Grayling (Thymallus thymallus (L.), T. nigrescens Dorogostaisky). Distribution: Europe (Russia, Yugoslavia), Asia (Russia, including Siberia, Mongolia). Reference: Scholz & Hanzelova´ (1998). Comments: Proteocephalus thymalli is a specific parasite of graylings and is almost indistinguishable morphologically from P. longicollis, which has been reported from a wide spectrum of salmoniform fish, including graylings (Freze, 1965; Dubinina, 1987; Scholz & Hanzelova´, 1998). Both taxa differ from each other only in the shape of the scolex, which is club-shaped in P. thymalli (versus rounded in P. longicollis – Scholz, Dra´bek & Hanzelova´ (1998) and in their susceptibility to experimental infection


(see Rusinek, 1987). Scholz & Hanzelova´ (1998) doubted the validity of P. thymalli but retained this species among the valid Proteocephalus species because they lacked suitable material for morphological and genetic evaluation. Rusinek (2001) described a new species, P. pronini, from the intestine of Thymallus nigrescens in the Mongolian Lake Hovsgol. The description is insufficient and some taxonomically important characters, such as the morphology of the cirrus-sac, the course of the vaginal canal, the presence/absence of a vaginal sphincter, the position of the seminal receptacle, etc., were not described or illustrated. Some other characteristics, such as the length of the neck and the number of proglottids and uterine diverticula, are also difficult to evaluate from the original description (Rusinek, 2001). The new species was differentiated from P. thymalli by only a few quantitative characteristics which exhibit high intraspecific and individual variability in the Palaearctic species of Proteocephalus (see Scholz & Hanzelova´, 1998 and references therein). In addition, measurements of the characters selected for the differentiation of P. pronini and P. thymalli presented in a comparative table (Rusinek, 2001) are almost identical or they overlap markedly. The most characteristic feature of P. pronini appears to be the presence of a clubshaped scolex with sublaterally situated suckers and a small, but distinct, flattened apical sucker (Rusinek, 2001 – Fig. A). However, a scolex of an identical shape is also the only morphological characteristic distinguishing P. thymalli from P. longicollis (see Hanzelova´ & Scholz, 1998; Scholz et al., 1998). Therefore, P. pronini is considered to be a junior synonym of P. thymalli, which occurs in conspecific fish hosts and has been found in the same region as P. pronini, i.e. northern Mongolia (Scholz & Ergens, 1990). Phylogenetic relationships: A comparison of the ITS2 sequences of P. thymalli with those of other species of Proteocephalus has revealed that it forms, together with P. ambiguus from Pungitius pungitius, a sister group of the clade composed of Proteocephalus longicollis and P. percae (Fig. 1). Sequence similarity between P. thymalli and P. longicollis is high (97.6-98.0% for the ITS2) and indicates that the validity of P. thymalli needs to be supported by new molecular data, because no PCR product for the V4 region of the 18S rRNA was obtained in this study.

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Rusinek & Kuznedelov (2002) published partial sequences (558 bp) of the 18S rDNA of two populations of P. thymalli from Russia and Mongolia, respectively. The sequences, which have not been deposited in GenBank and cover the highly conservative region at the 5’ end, were compared with those of other European Proteocephalus species to test their similarity and also possible synonymy with P. longicollis. The results showed a higher similarity of P. thymalli with P. percae (98.6%) and P. tetrastomus (98.2%) than with P. longicollis (96.8%). However, a closer study of the sequences of P. longicollis and P. thymalli showed inaccuracies at several positions which possibly affected the apparent similarity. Missing or redundant base at some conservative positions in P. thymalli could be caused by natural errors of the DNA polymerase. The most serious problem was in the variable region of the sequence of P. thymalli, in which non-standard F (?) bases were used (Rusinek & Kuznedelov, 2002), which did not permit more exact conclusions. Because there was no explanation for this symbol in the paper of the Russian authors, these bases were treated as unknown. Proteocephalus torulosus (Batsch, 1786) Syns Taenia torulosa Batsch, 1786; Proteocephalus ptychocheilus Faust, 1920; P. ruzskyi Titova, 1946; P. cobraeformis Haderlie, 1953 Type-host: Ide Leuciscus idus (L.) (Cypriniformes: Cyprinidae). Type-locality: Not known, probably Germany. Other hosts: Numerous species of cyprinid fish (e.g. Abramis, Alburnus, Barbus, Leuciscus, Phoxinus, Rutilus), with members of the Leuciscini being the most suitable hosts. Records from balitorid (Barbatula barbatula) and cobitid (Cobitis taenia) fish are questionable and need to be confirmed. Distribution: Europe, Asia (Siberia, Far East), western part of North America. References: Scholz & Hanzelova´ (1998, 1999). Comments: Recent taxonomic studies have shown that P. torulosus is a circumboreal parasite reported from numerous species of cyprinid fish (Scholz & Hanzelova´, 1998, 1999). It possesses some morphological characteristics unique or rare among the species of the Proteocephalus aggregate, such as a

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long, club-shaped scolex, with longitudinal wrinkles and without an apical sucker, testes arranged in at least two layers, a short cirrus-sac and a feebly developed vaginal sphincter (Scholz & Hanzelova´, 1998; Sˇkerˇ´ıkova´ et al., 2001). Phylogenetic relationships: A distinct position of P. torulosus among the taxa of the Proteocephalus aggregate, based on its morphology, is supported by the results of the phylogenetic analyses, in which this cestode forms a separate, most basal clade in almost all of the trees inferred from molecular data (Zehnder & Mariaux, 1999; Sˇkerˇ´ıkova´ et al., 2001, Hypsˇa et al., 2005; present data – Fig. 1). A separate position of P. torulosus is also reflected in the low values of sequence similarity with respect to other species of the Proteocephalus aggregate (Table 3).

Proteocephalus esocis (Schneider, 1905) – species inquirenda Syn. Ichthyotaenia esocis Schneider, 1905 Type-host: Pike Esox lucius L. (Esociformes: Esocidae). Type-locality: Reval, Estonia. Other host: Perca fluviatilis L. (Perciformes: Percidae). Distribution: Europe (Estonia, Russia). References: Anikieva (1995b), Scholz & Hanzelova´ (1998). Comments: This species was described by Schneider (1905) from the intestine of pike Esox lucius from Estonia and later also reported from Perca fluviatilis (Freze, 1965; Dubinina, 1987). Scholz & Hanzelova´ (1998), who studied the holotype of P. esocis, concluded that this species is apparently invalid and morphologically indistinguishable in its strobilar morphology from P. longicollis. However, because of the absence of the scolex in the type-specimens and the similarity between P. longicollis and P. percae in strobilar morphology, they did not synonymise P. esocis with either P. longicollis or P. percae. Anikieva (1995b) also considered Proteocephalus specimens from pike to belong to either P. exiguus (syn. of P. longicollis) or P. percae. The taxonomic status of P. esocis can be resolved only when a new material from the type-host and type-locality is available for morphological and genetic studies.

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Discussion The present list includes only 14 species of the Proteocephalus aggregate, which are considered to be valid, from fishes of the Palaearctic Region. This number is much lower than that reported in previous lists of proteocephalidean tapeworms. Freze (1965) reported 25 taxa, whereas Schmidt (1986) as many as 29 species. In addition, three other species, namely P. hemispherous Rahemo & El-Niaeemi, 2001, P. midoriensis Shimazu, 1990 and P. pronini Rusinek, 2001, have been described since 1986. So a marked difference in the number of valid species between individual authors seems to have been caused by several factors: (i) Freze (1965) and Schmidt (1986) included in their lists taxa described in the 19th Century, which had already been invalidated, e.g. by La Rue (1914); (ii) the books of these authors were largely compilative treatises rather than revisional monographs based on a critical evaluation of the type-material and voucher specimens (although Freze, 1965, redescribed some taxa occurring in Russia); (iii) in the 19th and the first half of the 20th Century, most species of Proteocephalus, including those of the Proteocephalus aggregate, were described on the basis of minor morphological differences of characters which exhibit great intraspecific and individual variability; the influence of many biotic and abiotic factors, especially the type and size of the definitive hosts, the intensity of infection, fixation methods, etc., on the morphology of individual species had not been considered by many authors; and (iv) genetic methods that may provide new diagnostic markers to distinguish morphologically uniform taxa have been applied to the systematics of the Proteocephalus aggregate only recently. A comparison of sequences of the nuclear genes (ITS2 and V4 region of 18S rRNA) enabled us to assess phylogenetic relationships of species of the Proteocephalus aggregate and also helped in a consideration of the validity of some morphologically similar taxa or species of doubtful validity. However, it should be pointed out that sequence differences between some taxa were very low, even lower than between two isolates of P. longicollis from different hosts and geographical regions (Table 3). Although this species is polymorphic (Hanzelova´, Scholz, & Fagerholm, 1995; Hanzelova´, Sˇpakulova´, Fagerholm, et al., 1995; Scholz & Hanzelova´, 1998), more


molecular data are necessary to confirm its validity or to detect the possible existence of cryptic species, as well as to better assess the phylogenetic relationships of some taxa, especially those for which sequences of only one gene, either ITS2 or 18S, were available, i.e. P. gobiorum and P. thymalli. Notwithstanding the above-mentioned caution regarding the genetic delimitation of some taxa, the systematics of this group of fish tapeworms is better resolved than that of many other proteocephalidean cestodes as a result of multidisciplinary studies carried out recently (see Scholz & Hanzelova´, 1998; Scholz & de Chambrier, 2003; Hypsˇa et al., 2005, for references). This is also valid for presumed congeneric species found in the Nearctic Region. Regarding the number of species of Proteocephalus (their assignment to the Proteocephalus aggregate requires further systematic studies – Scholz & de Chambrier, 2003) from freshwater fishes from North America (without Neotropical Mexico and Cuba), Freze (1965) and Schmidt (1986) listed as many as 31 and 33 nominotypical taxa, respectively. Taking into account the narrow host-specificity of most Proteocephalus species in the Palaearctic Region, i.e. those of the Proteocephalus aggregate (Scholz & Hanzelova´, 1998), it is questionable that some North American fish, such as largemouth bass Micropterus salmoides, can be infected with as many as 10 species of Proteocephalus (Freze, 1965; Hoffman, 1999). It is probable that numerous taxa described from North American fishes are synonymous and the actual number of valid species is much lower, as found for the Palaearctic Region. Indeed, a taxonomic study of Hanzelova´ & Scholz (1999) has demonstrated that most probably only one species, P. longicollis, rather than 11 and 13 as listed by Schmidt (1986) and Hoffman (1999), respectively, occurs in salmonid fishes from North America. Another study of the same authors (Scholz & Hanzelova´, 1999) has also revealed that P. ptychocheilus Faust, 1920 and P. cobraeformisHaderlie, 1953, described from cyprinid fishes in the western part of North America, are conspecific with P. torulosus, which has a Holarctic (circumpolar) distribution. Of the species of the Proteocephalus aggregate found in the Palaearctic Region, the following taxa have a circumpolar distribution: P. filicollis, P. fluviatilis (apparently of Nearctic origin, introduced to Japan with large- and smallmouth

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bass), P. longicollis, P. macrocephalus, P. tetrastomus and P. torulosus. Another species of Proteocephalus, P. parasiluri Yamaguti, 1934, was described from Silurus asotus L. in Japan (Yamaguti, 1934). It possesses a metascolex and was placed in Paraproteocephalus Chen in Dubinina, 1962 by Shimazu (1993). Proteocephalus also contained another species considered to be valid by Scholz & Hanzelova´ (1998), namely P. osculatus (Goeze, 1782) (syns Taenia osculata Goeze, 1782; Ichthyotaenia skorikowi von Linstow, 1904). It is a specific parasite of the European wels Silurus glanis L. in Europe, including Russia, and Asia (Azerbaijan, Iraq, Central Asia), and also occurs accidentally in sturgeons (Acipenser stellatus Pallas, Pseudoscaphirhynchus kaufmanni (Kessler)) in the Caspian Sea (Anikieva & Kharin, 1997; Scholz & Hanzelova´, 1998; Skryabina, 1974). de Chambrier et al. (2004) proposed a new genus, Glanitaenia, to accommodate Proteocephalus osculatus, because in all molecular analyses it formed a clade with Paraproteocephalus parasiluri (Yamaguti, 1934) Shimazu, 1993 [nec P. parasiluri (Zmeev, 1936) Chen in Dubinina, 1962], a species typically possessing a metascolex (Shimazu, 1993), thus making the Palaearctic species of Proteocephalus paraphyletic. Glanitaenia osculata is also markedly distinct from Proteocephalus species in possessing a well-developed, functional apical sucker and a greater number of testes. Recent data of Hypsˇa et al. (2005) and those of the present study fully support the validity of Glanitaenia. Rahemo and Al-Niaeemi (2001) described Proteocephalus hemispherous from the intestine of Silurus glanis in the River Tigris at Mosul, Iraq. They distinguished it from P. osculatus (=G. osculata) in possessing a large and well-developed apical organ, nearly square mature proglottids and the shape of the ovary. Conspecific tapeworms were misidentified as Silurotaenia siluri (Batsch, 1786) by Ali, Al-Jafery, and Abdul-Ameer (1987), Al-Kalak (1992) and Mohamad (1995), despite the absence of any spines on the apical organ. P. hemispherous is undoubtedly a member of the Gangesiinae, based on the morphology of the scolex and strobila. It may well be conspecific with Postgangesia inarmata de Chambrier, Al-Kallak & Mariaux, 2003, described from the same fish host (S. glanis) in Iraq. However, there is a marked diference in the number of testes between these taxa (70-80 in P. hemispherous versus 115–151 in Po. inarmata)

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and the apical organ of P. hemispherous appears to be much deeper than that of P. inarmata, in which it is flattened (de Chambrier et al., 2003; Rahemo & AlNiaeemi, 2001). Therefore, P. hemispherous is transferred to Postgangesia as Postgangesia hemispherous (Rahemo & Al-Niaeemi, 2001) n. comb. Its possible conspecificity with P. inarmata should be verified on the basis of comparison of the type or voucher specimens of both the taxa. Recent phylogenetic studies (de Chambrier et al., 2004; Hypsˇa et al., 2005; Sˇkerˇ´ıkova´ et al., 2001; Zehnder & Mariaux, 1999; present data) provide evidence for the monophyly of the Palaearctic species of Proteocephalus, provided that G. osculata is excluded from Proteocephalus, as proposed by de Chambrier et al. (2004). These authors proposed this group to form the Proteocephalus aggregate in order to distinguish it from other, apparently unrelated members of the composite genus Proteocephalus. Monophyly of the Proteocephalus aggregate corresponds well with a rather uniform morphology of its members, compared to the extreme morphological variation of members of Proteocephalus (sensu lato) occurring in the Neotropical Region (see de Chambrier & Vaucher, 1999; Rego et al., 1998). Regarding evolutionary relationships with their fish hosts, existing data suggest an incongruence between the phylogenies of the species of the Proteocephalus aggregate and the fish they infect, thus indicating host-switching events rather than co-evolution, as already claimed by Sˇkerˇ´ıkova´ et al. (2001). Based on the position of individual species in the phylogenetic tree, it is obvious that some morphological characters may be homoplastic and may have appeared independently during the evolution of the group. For example, the absence of an apical sucker in P. gobiorum is apparently a derived character, because this species appears in the clade containing species with a distinct apical sucker (P. filicollis, P. fluviatilis and P. macrocephalus), whereas other taxa are devoid of an apical sucker (P. torulosus, P. sagittusand P. midoriensis) and belong to unrelated, more basal clades (Fig. 1). On the contrary, Glanitaenia osculata (syn. Proteocephalus osculatus), which is basal to all species of the Proteocephalus aggregate, including P. torulosus (see Hypsˇa et al., 2005; this study), possesses a well-developed apical sucker with a deep cavity. However, the least reduced apical sucker is present in P. longicollis,

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P. thymalli and P. percae, which together form the most derived clade in the tree (Fig. 1). In order to facilitate identification of tapeworms of the Proteocephalus aggregate sensu de Chambrier et al. (2004) considered to be valid, the following key is provided.

Key to species of the Proteocephalus aggregate from fishes of the Palaearctic Region This key is primarily based on morphological features of the scolex and strobila summarised by Shimazu (1990 – species from Japan) and Scholz and Hanzelova´ (1998 – species from Europe, including Russia). However, the spectrum of definitive hosts is also used as a differential criterion because some valid taxa occurring in unrelated fish hosts differ only slightly in their morphology. If possible, only one or a few differential characters are provided in the key. More data on the morphology of individual taxa can be found in their redescriptions (Rødland, 1983; Shimazu, 1990; Scholz & Hanzelova´, 1998; Scholz, Dra´bek & Hanzelova´, 1998; Scholz et al., 2003). 1.

– 2. – 3.

– 4.

– 5.

Proglottids of markedly trapeziform shape; immature proglottides short and very wide. Apical sucker reduced, difficult to see. In smelt (Osmeridae). ………………….. P. tetrastomus Proglottids of rectangular shape ……………. 2 Scolex with apical sucker …………………... 3 Scolex without apical sucker ……………... 11 Scolex terminally blunt, with small apical sucker. Cirrus-sac short, only its proximal third crosses vitelline follicles; vaginal sphincter absent. In ruff (Gymnocephalus) …………………………………….. P. cernuae Scolex of different shape ………………….. 4 Large tapeworms (up to 200-300 mm in length), with relatively short cirrus-sac, representing about 1/8-1/4 of proglottid width ………………………………………………. 5 Small tapeworms or cirrus-sac longer, about 1/3-2/5 of proglottid width ……..…………... 6 Apical sucker vestigial, longer than wide. Vaginal canal widened, thick-walled distally; seminal receptacle far anterior to ovarian isthmus. In eels (Anguilla) ……………………………... P. macrocephalus


– Apical sucker vestigial, widely oval. Vaginal canal without prominent thickening in distal part; seminal receptacle just anterodorsal to ovarian isthmus. In centrarchids (Micropterus) ………………………………….... P. fluviatilis 6. Vaginal sphincter well developed; cirrus-sac elongate, long, representing 1/3-2/5 of proglottid width …………………………………….. 7 – Vaginal sphincter absent; cirrus-sac pyriform, short, representing less than 1/3 of proglottid width. In sticklebacks (Gasterosteidae) …………………………………………….... 10 7. Scolex tapering anteriorly; neck indistinct, wider than scolex. In perch (Perca fluviatilis) and other percid fishes (Percidae)……P. percae – Scolex not tapering anteriorly; neck usually distinct, narrower than scolex …..………….. 8 8. Scolex club-shaped, with neck far posterior to suckers. In grayling (Thymallidae) ……………………………………...P. thymalli – Scolex small, rounded; neck close posterior to suckers………………………………………. 9 9. Apical sucker large, 22-86 mm in diameter. In salmoniform fishes, rarely in smelt (Osmeridae) ………………………………….. P. longicollis – Apical sucker small, 4-7 mm in diameter. In ayu (Plecoglossus) .………………... P. plecoglossi 10. Strobila